PROJECT SUMMARY/ABSTRACT Human T cells are central to physiological immune homeostasis, which protects us from pathogens without collateral autoimmune inflammation. Polyclonal populations of T cells have been used as cancer therapies (tumor-infiltrating lymphocytes), and recently polyclonal regulatory T cells (Tregs) in type 1 diabets (T1D). However the vast majority of polyclonal T cells do not recognize a desired tumor specific or auto-antigen. Engineering antigen specificity by introduction of a new T cell receptor (TCR) or chimeric antigen receptor (CARs) makes the transferred cells much more potent, shown in human cancer trials and in mouse models of type 1 diabetes. Such engineering can be accomplished by using retroviral vectors, and recently genome editing has brought the promise of specific and efficient insertion of large transgenes. However these approaches still require viral transduction, slowing research and clinical use. To overcome these limitations, in my preliminary work I have developed a novel non-viral, CRISPR-Cas9 genome targeting system that permits the rapid and efficient insertion of individual or multiplexed large (>1 kilobase) DNA sequences at specific sites in the genomes of primary human T cells while preserving cell viability and function. In my first aim, I propose to use this non-viral genome targeting system to replace the endogenous TCR. This approach will redirect a therapeutic T cell's antigenic specificity while maintaining its endogenous TCR expression and minimizing TCR mispairing. For my second aim, I will show that replacement of regulatory T cell's endogenous TCR with a T1D autoantigen specific TCR using non-viral gene targeting will create a more potent and clinically viable cellular therapeutic for T1D. I will demonstrate the ability to redirect both mouse and human regulatory T cells to recognize a defined T1D autoantigen and assay their in vitro and in vivo functionality in preventing and reversing T1D development. My sponsor Dr. Alex Marson has extensive expertise in the genetic basis of T1D and the genetic engineering of primary T cells; my co-sponsor Dr. Mark Anderson has made foundational discoveries about the nature of immune tolerance in T1D. In addition to my two sponsors, my ongoing local collaborations with Dr. Jeff Bluestone (leading the first clinical trials of polyclonal regulatory T cells in human T1D patients) and Dr. Kole Roybal (applying synthetic biology to re-engineer T cell specificity) will further support the feasibility of the proposed work. Similarly, I am undergoing longitudinal clinical training in cellular therapeutics with Dr. Jonathan Esensten, the medical director of UCSF's Regulatory T Cell Therapy Group. Overall, this work will lay the foundation for clinical application of non-viral TCR replacement in regulatory T cells as a curative cellular therapy for T1D.